Rhamnogalacturonan-II (RG-II) is a low molecular weight, structurally well-defined, complex pectic polysaccharide that is released from the walls of plant cells by treatment with endo-.alpha.-1,4-polygalacturonase York et al. (1985) Methods Enzymol. 118:3-40!. RG-II has also been isolated from the cell walls of many plant cells, for example, sycamore (Acer pseudoplatanus) Darvill et al. (1980) in The Plant Cell (N. E. Tolbert, ed.) Academic Press, New York, pp. 91-162!, Douglas fir (Pseudotsuga menziesii) Thomas et al. (1987) Plant Physiol. 83:659-671!, rice (Oryza sativa) Thomas et al. (1989) Carbohydr. Res. 185:261-277!, onion (Allium cepa) Stevenson et al. (1988) Carbohydr. Res. 179:269-288!, kiwi fruit (Actinidia deliciosa) Redgwell et al. (1991) Carbohydr. Res. 209:191-202!, etc., and is present in the medium of cultured sycamore cells Stevenson et al. (1986) Plant Physiol. 80:1012-1019!, in the commercial enzyme preparation Pectinol AC Spellman et al. (1983) Carbohydr. Res. 122:115-129!, and in red wine Doco et al. (1993) Carbohydr. Res. 243:333-343!.
RG-II contains eleven different glycosyl residues O'Neill et al. (1990) Methods Plant Biochem. 2:415-441!, including the unusual monosaccharides apiose, 3-C-carboxy-5-deoxy-L-xylose (aceric acid), 3-deoxy-D-manno-octulosonic acid (Kdo), and 3-deoxy-D-lyxo-heptulosaric acid (Dha). RG-II also contains the seldom observed methyl-etherified sugars 2-O-methyl xylose and 2-O-methyl fucose. Many of the glycosidic linkages and ring forms of the glycosyl residues of RG-II, including .beta.-D-galactosyluronic acid, .beta.-L-arabinofuranose, .alpha.-L-arabinopyranose, and a fully substituted rhamnosyl residue, are also unusual. Some of the glycosyl residues in RG-II are O-acetylated. The backbone of RG-II has been shown to be composed of at least seven 1,4-linked .alpha.-D-galactosyluronic acid residues Puvanesarajah et el. (1991) Carbohydr. Res. 218:211-222!, schematically presented in FIG. 1.
A variety of oligosaccharide side chains are attached to the backbone Stevenson et al. (1988) Carbohydr. Res. 182:207-226!. The carboxyl groups of some of the D-galactopyranosyluronic acid residues are esterified with methyl groups Puvanesarajah et al. (1991) Carbohydr. Res. 218:211-222!. Partial acid hydrolysis of both native and O-permethylated RG-II have led to the isolation and structural characterization of oligosaccharide fragments of RG-II containing its glycosyl residues York et al. (1985) Carbohydr. Res. 138:109-126; Stevenson et al. (1988) Carbohydr. Res. 182:207-226; Stevenson et al. (1988) Carbohydr. Res. 179:269-288; Puvanesarajah et al. (1991) Carbohydr. Res. 218:211-222; Spellman et al. (1983) Carbohydr. Res. 122:131-153; Melton et al. (1986) Carbohydr. Res. 146:279-305!. RG-II (see FIG. 1) has been shown to have the same structure in every plant from which it has been isolated.
RG-II isolated from radish roots Kobayashi et al. (1996) Plant Physiol. 110:1017-1020; Matoh et al. (1993) Plant Cell. Physiol. 34:639-642! and from sugar beet Ishii et al. (1996) Carbohydr. Res. 284:1-9! were found to contain borate esters. Boron is known to be an essential microelement for normal plant growth and development Loomis et al. (1992) BioFactors 3:229-239; Hu et al. (1994) Plant Physiol. 105:681-689; Shelp et al. (1995) Physiol. Plant 94:356-361; Welch (1995) Crit. Rev. Plant Sci. 14:49-82!. Boron deficiency, which first becomes apparent in growing tissues, results in disorganized cell expansion and the formation of cell walls with abnormal morphology Loomis et al. (1992) BioFactors 3:229-239!. Growing plant cells require a constant supply of exogenous boron because the majority of boron in plant tissues is present in a "nonavailable" form Loomis et al. (1992) BioFactors 3:229-239; Hu et al. (1994) Plant Physiol. 105:681-689; Brown et al. (1994) Physiol. Plant 91:435-441!. Boron is believed to form borate-diol esters that covalently crosslink cell wall pectic polysaccharides (Kobayashi et al. (1996) Plant Physiol. 110:1017-1020; Matoh et al. (1993) Plant Cell Physiol. 34:639-642; Ishii et al. (1996) Carbohydr. Res. 284:1-9!. Also, borate ester cross-linking of polysaccharides in vitro has been shown to be pH-dependent Deuel et al. (1954) in Natural Plant Hydrocolloids Adv. Chem. Series No. 11! pp. 51-61, American Chem. Society, Washington, D.C.!. Thus, it has been suggested that boron cross-links are the "load-bearing", acid-labile linkages that are hydrolyzed by a decrease in cell wall pH during auxin-induced cell expansion Loomis et al. (1992) BioFactors 3:229-239!. RG-II is the only boron-containing polysaccharide that has been isolated from a biological source.
Contamination of the environment with heavy metal ions and/or alkyl and thiol derivatives of metals has increased over the last several decades, with toxic levels of the contaminants being reached in air, water and/or soil in certain locations. Contamination may stem from human and industrial sources. Heavy metals are an increasing problem in the sludge produced by industries and populations centers Adriano (1986) "Trace elements in the terrestrial environment," New York, Springer Verlag; Alloway (1990) "Heavy metals in soils" New York, John Wiley & Sons!. The wind borne residue of volatile metals have contaminated land at great distances from smelting operations, rendering it useless Lepp (1981) "Effects of heavy metal pollution on plants" In Appl. Sci. Editor, Applied Science Publishers, New Jersey 12!. Land in some parts of the globe such as the western United States Cannon (1960) "The development of botanical methods of prospecting for uranium on the Colorado Plateau," U.S. Geol. Surv. Bull. 1085A:1-50!, and Africa Brooks and Malaisse (1985) The Heavy Metal-tolerant Flora of South Central Africa, A. A. Balkema Press, Boston, Mass.! is naturally contaminated with high levels of a variety of toxic metals including arsenic, cadmium, copper, cobalt, lead, mercury, selenium and/or zinc. A major concern is the ability to dispose of wastes containing toxic heavy metals generated by weapons production facilities, power generation plants, mining and the metal fabrication industries. Stabilizing and reducing the mass of the toxic metals contained in such wastes would facilitate their disposal.
These heavy metals are often found in soil and marine sediments as heavy metal salts (e.g., thiol salts), as chelates with acidic humic substances (for example, methylmercury), and to a lesser extent other organocationic species, and as free multivalent cations. Some heavy metals cycle through the aqueous phase and into the atmosphere as volatile elemental cations, free or complexed, and are then oxidized and washed by rain into the marine environment Barkay et al. (1992) Biodegradation 3:147-159!. Some bacteria in soil and sediments can detoxify heavy metal cations by reducing them to their metallic forms. Heavy metals are often found bound in the form of organocomplexes in contaminated animals and microbes Barkay et al. (1992) supra; Robinson and Tuovinen (1984) Microbiological Reviews 48:95-124!. In fish, where heavy metal toxicity is well studied, most of the tissue-associated heavy metals are found as organocomplexes Pan Hou and Imura (1987) Arch. Microbiol. 131:176-177!. Heavy metal organocomplexes may be volatile and extremely toxic to plants and, generally, to the environment D'Itri and D'Itri (1987) Environ. Management 2:3-16!.
With global heavy metal contamination on the increase Nriagu and Pacyna (1988) Nature 333:134-139!, plants which can process heavy metals might provide efficient and ecologically sound solutions. Regions which are naturally contaminated with heavy metals are often characterized by scrubby heavy-metal tolerant vegetation Brooks and Malaisse (1985) The Heavy Metal-tolerant Flora of South Central Africa, A. A. Balkema Press, Boston, Mass.; Wild, H. (1978) "The Vegetation of Heavy Metal and Other Toxic Soils," in Biogeography and Ecology of Southern Africa, Wergren, M. J. H., ed., Junk, The Hague, Netherlands!. Certain of these naturally occurring metal-resistant plants hyperaccumulate large amounts of heavy metals in the form of malate or citrate chelates. These plants have been found in a variety of habitats, but often they exhibit bizarre metal ion requirements, grow poorly in less exotic habitats, and are of little direct economic value as crop or forest species.
There is a long felt need in the art for the in situ detection and detoxification of toxic heavy metal ions and/or heavy metal complexes in human and animal applications and in their environment. The present invention provides an assay to detect specific heavy metal cations and a method to complex such, and enables phytoremediation and/or revegetation of contaminated environments via plants and plant components capable of heavy metal cation complexation and sequestration.